Liquid-Liquid Extraction

also known as distribution chromatography or liquid-liquid chromatography.

The separation of substance mixtures is often very difficult due to poor sample solubility, composition of the ingredients and their large differences in polarity. Based on solid-liquid extraction the classical chromatographic methods such as HPLC, MPLC or Flash, show considerable disadvantages when used in applications with complex mixtures such as natural product extracts or synthesis products. In addition to the time-consuming and costly processing of such a mixture, the up-scaling of established methods is not self-evident. Here, the liquid-liquid extraction offers considerable advantages. Due to its liquid stationary phase, the ingredients are only separated by their respective polarity, i.e. according to their distribution coefficient KD. In practice, the distribution chromatography is realized with the SCPC.

Key Features

  • No irreversible adsorption of sample components to a solid carrier guaranteeing 100% sample ingredient recovery
  • Little or no sample preparation.
  • Fractionation of the mobile phase and liquid stationary phase.
  • High loading capacity ensuring direct up-scaling from milligrams to a kilogram scale.
  • No sample solubility limit because of large solvent volumes and solution in polar and non-polar phase.
  • Switching from mobile to stationary phase during the chromatography run.
  • Great savings in time and solvents.

The Continuous Distribution

The continuous distribution in the liquid-liquid extraction is obtained by the serial connection of individual separation sites, called chambers. When all the chambers are filled with both phases of a 2-phase mixture, for example n-BuOH/H20, one of these phases (mobile phase) will be transported through the chamber system. The other phase (stationary phase) is retained by centrifugal force in the chamber system thus rendering chromatography without solid column material possible. Analogous to the static distribution you obtained here K=CSP/CMP with CSP as the concentration of a substance in the stationary phase and CMP as the concentration of the same substance in the mobile phase.


The separation unit of the SCPC is the rotor, with a separating system of interconnected chambers. After filling of the rotor with the liquid stationary phase and the beginning rotation, the mobile phase is pumped through the rotating rotor. The mobile phase replaces a portion of the stationary phase and after equilibration the sample application can be carried out.

During the chromatographic run the individual fractions segregate with different velocity to the chamber system outlet according to their different distribution coefficients K. The high resolution of the sample constituents in the SCPC is achieved by the large number of chambers and the good mass transfer between the two phases.

Inside the rotor the rotor chambers, interconnected in series, function as hydrostatically working chromatographic column (separating funnel principle). After passing through over 1000 chambers with non-miscible phases the sample ingredients exit the rotor separately.



SCPC Rotor


SCPC Rotor chambers

Rotor chambers

SCPC Distribution in the chambers

Distribution in the chambers

Simple Substance Isolation

Classic chromatographic separation methods such as HPLC, MPLC, Flash, etc. work with solid column material as the separation medium, making the separation of natural extracts from plants, soil and fungi difficult. On the one hand occurring matrix effects caused by chlorophylls, humic acids, mucilage, etc. impede the separation and on the other the life span of the separating columns is reduced.

Here, sample pretreatment plays an important role in the overall preparation process. The same is true for organic syntheses and the separation of its by-products from the target product. Distribution chromatography, in contrast, allows a problem-free and fast processing of such samples. The liquid 2-phase system usually makes sample pretreatment obsolete or limits it to sample dilution.

The distribution chromatographic method applied with the SCPC works absolutely loss free as the absence of a solid phase makes irreversible adsorptions of sample components impossible. There is no interference by the usual matrix effects or poor sample solubility, because the sample can be injected dissolved in both of the two phases. This is favorable especially when sample constituents show opposing polarity.

The technically simple method of liquid-liquid extraction renders substances separation of complex mixtures possible quickly and efficiently. Applications range from antibiotics, steroids, peptides, proteins to phenols, polyphenols, terpenes, glycosides, saponins, flavonoids, alkaloids, plant hormones, insecticides, herbicides, fatty acids and sugars. Special phase mixtures even allow the separation of metals, isomers and chiral samples. In addition to the separation of ingredients, the distribution chromatography is also ideal for the accumulation of minimum quantities. Examples for this application are surfactant measurements from surface waters or determination of pollution in soils. Given the possibility of linear upscaling separations up to a kilogram scale can easily be performed.

The flow rate is independent of the sample amount and only dependent on the rotor volume. The flow rate for liquid-liquid chromatography corresponds to approximately 1/10 of the flow rate required for the purification of a comparable sample amount by HPLC. This immensely reduces solvent consumption in preparative chromatography and thereby also reduces the over-all costs, especially since solvents of inferior quality can be used.

The following examples of use show some practice-oriented applications of the liquid-liquid extraction. Developed in the 1940s of the last century by Dr. Craig, liquid-liquid extraction was refined to an equal separation technology among other chromatographic methods.

Application Example 1

Isolation of 10-deacetylbaccatin III (10-DAB) from the needles of the Taxus chinensis plant.

HPLC Chromatogram with SCPC 1

[Fig. 1] HPLC chromatogram of the crude extract of Taxus chinensis.

HPLC Chromatogram with SCPC 2

[Fig. 2] HPLC chromatogram of 10-DAB Group and SCPC chromatogram of the crude extract of Taxus chinensis with 10-DAB peak. Sample size 1,000 mg crude extract; Solvent system: n-hexane/chloroform/MeOH/water; SCPC with 200 ml rotor.

The method shows a fast semi-preparative purification of the target fraction 10-DAB from the crude extract, followed by HPLC analysis of the target fraction. In almost the same chromatography time liquid-liquid extraction with the SCPC only needed a total of about 1 liter of solvent in p.a. grade. The HPLC, in contrast, you would have to run with a 5 cm ID column that would consume about 4 liters of solvent in HPLC-grade. For liquid-liquid extraction the crude extract could be injected directly without sample pretreatment. Taking all cost causative factors into account, the SCPC only reaches 15-20% of the costs entailed by HPLC.

Application Example 2

Isolation of a target substance from the root extract of a South African plant.

HPLC Chromatogram with SCPC 3

[Fig. 3] Indicative of natural materials the analytical HPLC chromatogram of the crude extract shows a variety of unknown substances (peaks). A method development for the isolation of the target substance (arrow) on the basis of analytical data is very time consuming. The target substance is present only in the range of a few percent. The purification via SCPC is recommended.

HPLC Chromatogram with SCPC 4

[Fig. 4] HPLC chromatogram of the accumulated extract with target substance (arrow). In order to achieve efficient isolation results and extract the target substance, the target substance from the root extract was accumulated by a single extraction step to over 20%.

HPLC Chromatogram with SCPC 5

[Fig. 5 left] The enriched extract, peak 3, after separation by the SCPC with typical peak shapes of liquid-liquid extraction.
[Fig. 5 right] HPLC chromatogram of the target fraction peak 3 with 98% purity.

Solvent system: heptane/ethyl acetate/ethanol/water. Two days Method development for the distribution chromatography.

Application Example 3

For the separation of chiral substances on a preparative scale liquid-liquid extraction is the established cost effective alternative to HPLC. From a great variety of chiral selectors, however, only four are practically used, one example is mentioned here.

HPLC Chromatogram with SCPC 6

[Fig. 6] Separation of 4 DNB-amino acid racemates by the SCPC. N-dodecanoyl-L-proline-3,5-dimethyl aniline as chiral selector. Solvent system: hexane/EE/MeOH/10mM HCL, amount of substance 10 mg per DNB – amino acid [10]. The chromatograms show the amino acid sample with and without chiral selector.

A very effective method for the separation of polar compounds is the pH-zone refining. By adding e.g. TFA to the organic phase and e.g. NH3 to the aqueous phase a pH-gradient is generated which, because of the different pKs-values of the substances, leads to separation of the polar substances with clear-cut distinction of the sample constituents.

HPLC Chromatogram with SCPC 7

[Fig. 7]

The chromatogram shows a separation of 8 amino acids. The pH-zone refining is tailored exactly to the separation of polar substances. Since the mobile phase is constantly exchanged with the stationary phase during the entire chromatography time, the pH gradient extends over the entire length of the rotor.

Solvent system: MTBE/ACN/H2O, substance quantities of 100 mg per peptide.


The technical realization of the continuous distribution has made the liquid-liquid extraction to an inexpensive, fast and above all flexible method for separating even the most difficult samples.

The flexibility is based on a great variety of solvent systems, the advantages of a liquid stationary phase, and simple processing of samples with difficult matrix effects. The SCPC alone or in combination with HPLC leads to accelerated purification of crude extracts and synthesis products and compared to merely HPLC based separation methods a time reduction of up to 10 fold can be observed. A further advantage of distribution chromatography is the direct upscaling of liquid-liquid extraction methods to a kilogram scale. This type of rapid substance extraction is of great benefit for subsequent methods such as metabolite determination, structure elucidation, and many more. The great amount of papers published worldwide shows that the distribution chromatography is about to become a standard method in addition to HPLC and DC in chemistry, pharmaceutical chemistry, pharmaceutical biology, chemical ecology and other scientific fields.

History of Distribution Chromatography, also Known as Liquid-Liquid Extraction

The quest for identifying the components of material things, their resolution and the separation of substances was a challenge for human kind at all times.

Separating Funnel

Separating Funnel

To this day the traditional way of extracting substances from plants, soil or food with the aid of water in a separating funnel (SF) is used. This procedure has been improved by the addition of water-immiscible solvents, thereby obtaining a liquid 2-phase system.

Sample components injected into this system then segregated to either of the two phases. This was the first step of a separation process of substances or compounds.

Craig Principle

Craig Principle

In 1940 the scientist Dr. Craig improved the separation efficiency by the serial interconnection of glass vessels. These so-called Craig-systems are sometimes still used today for the purification of peptides. The disadvantages of these systems are the great solvent consumption and the large dimensions of the plant.



In order to reduce system size and solvent consumption various devices were placed on the market over the years, from 1960 to 1980, basing on the CCC separation technology (Counter Current Chromatography). This technology uses the physical effect of separating the sample components by their differences in polarity in a 2-phase solvent system.



A first test-unit according to Craig was the DCCC (Droplet Counter Current Chromatograph) with long separation times, low sample throughput and leakage problems. The same fate of low acceptance in the market befell the RLCCC (Rotary Locular Counter Current Chromatograph). Constant leakage problems, long filling times and limitations in the applicable sample amounts made the RLCCC disappeared from the market quickly.



In the late 80s, better separation results and acceptable chromatography times were realized by the HSCCC (High Speed Coil Counter Current Chromatograph) via liquid-liquid extraction. Because of its technical design, the planetary suspension, only small sample amounts of 2 to 3 g could be separated in a single run.



A first attempt to use the CCC-technology in the industry was undertaken 1991 with the introduction of LSCCC (Low Speed Coil Counter Current Chromatograph). Here, as well, production of the device stopped after a few years because of the mismatch of extensive space requirements and high weight of 300 kg with only 12-15 g sample amount per run. Comparable to the HSCCC, the planetary suspension of the LSCCC was the greatest impediment for upscaling and acceptable industrial separation times.



The breakthrough in the liquid-liquid extraction was achieved in 1997 with the development of the SCPCen, because the SCPC provides very rapid purifications in a very large sample scale. For this optimized separation technology not glass vessels, but rather over a thousand interconnected chambers in metal plates are used as separation sites. Many of these stacked metal disks ad up to a rotor whose rotational speed is electronically controlled in the SCPC. The desired separations take place in the rotor chambers by centrifugal force in the liquid stationary phase and in the liquid mobile phase.

In practice, the SCPC is attached to the HPLC instead of the HPLC column with solid carrier, virtually as a rotating column. Also the combination of both is best suited for e.g. many purifications of problematic substances. This combination allows the SCPC to function as a first purification step followed by a fine purification of the pre-cleaned substance via the HPLC solid phase column in the same run.

This method has the great advantage of reduced solid phase column clogging and following replacement, moreover, much solvent can be saved. Due to its versatility, speed and many other advantages the SCPC is becoming increasingly common in the areas of research, development and production.